The goal of this Phase II grant is to expand the Phase I demonstration of improvements to protein crystallization achieved using engineered nucleation surfaces to other high impact targets that include proteins involved in antibiotic resistance and membrane proteins. The role of crystallography in establishing the structure of biological macromolecules plays a key role in understanding their function, and Public Health benefits will derive from more efficient targeted drug development. Crystallization of proteins remains a challenge, and current approaches are experimentally intensive and have >75% failure rates, which creates a ?crystallization bottleneck? that is the rate limiting step in structural biology. Phase I Aims were achieved using surface energy modificationslargely without the need for microdomain thermal perturbations. Results showed an average reduction in crystallization onset times of 31% with a 24 fold increase in the number of crystals/experiment, of which 74% formed on the engineered nucleation features, for bovine pancreatic trypsin, thaumatin, lysozyme, and ?-lactoglobulin (n=15?26 each). DeNovX?s products will be 384 and 96 well HTS plates for crystallization, and this slotin strategy fosters technology adoption by fitting into existing workflows and equipment. The innovative aspects of the patented platform technologies are surface modifications that give tunable surface energies that enhance crystal nucleation. Our hypothesis is that success outcomes for difficult to crystallize proteins can be improved using surface energy modifications to improve crystal nucleation. Specific Aim 1 ? Expand the ongoing probe of crystallization function to at least 20 protein targets with an emphasis on those involved in the emerging threats from antibiotic resistance (e.g., ?-lactamases, etc.,) via a subaward to Argonne National Laboratory and the Center for Structural Genomics of Infectious Diseases. Specific Aim 2 - Initiate systematic investigations that advance the mechanistic understanding of how different surface energy modifications improve crystal nucleation for various nucleation site characteristics using 810 diverse proteins of 1070 kDa. This understanding will pave the way for rational tailoring of the surface energy modifications for use with other soluble proteins and with 2-3 membrane proteins. Specific Aim 3 - Expand end user research and prototyping to ensure HTS slotin compatibility for soluble protein applications. Initiate ??site testing making HTS formats with surface energy nucleation enhancements available for use in membrane protein facilities controlling these precious and perishable assets. A better functional and mechanistic understanding of how surface energy modifications enhance protein nucleation is expected to improve crystallization outcomes for proteins recalcitrant to crystallization. DeNovX?s innovative, scientifically rigorous, and robust approach to improving crystal nucleation serves as the foundation for a high value life sciences consumable product line.